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Assignment 5: Methods in Genetics and Genomics--Polymerase Chain Reaction (PCR)
Overview, History & Uses of PCR: Polymerase Chain Reaction, or PCR, is a form of biotechnology that allows a scientist to amplify a single piece or several pieces of DNA, generating thousands to millions copies of the desired DNA sequence. PCR is made possible by "thermal cycling," where the DNA target is repeatedly heated and cooled. Primers (short DNA fragments that here contain sequences complementary to the target region) and DNA polymerase are two essential components of PCR. The DNA generated by PCR is itself used as a template for replication, which basically translates into a chain reaction where the DNA template is amplified exponentially. PCR was created by Kary Mullis in 1983, and truly revolutionized the way in which DNA could be used in the lab and for medical research (Mullis won a Nobel Prize for his invention). Prior to PCR, laboratory DNA replication techniques were incredibly costly, elaborate and time-consuming. Mullis' technique both simplified and made more accessible the process of DNA amplification for a wide variety of uses. Furthermore, nothing prior to PCR had ever been so efficient in replicating a sequence of DNA, as the entire procedure from start to finish takes under an hour. Use of PCR in Genetics and Genomics: Since its inception, PCR has become one of the most widely-used techniques in molecular biology and genomics. PCR requires a minimum of reagents and equipment compared to other similar lab procedures, and can be set up simply and quickly. It can be as useful to the "solo underfunded bench scientist as it is to teams of production scientists using the most advanced automated equipment, generating significant amounts of data in a short time." The main reason for PCR's unprecedented impact on the lab science arena is its ability to detect and amplify as little as a single copy of a particular nucleotide sequence. Moreover, PCR can be used for the quantification of specific sequences "because of the quantitative relationship between the amount of starting target sequence and the amount of PCR product at any given cycle that falls within the reaction's exponential range." In other words, PCR is a technique that has both qualitative (identification) and quantitative capabilities for replicating a sequence of DNA or identifying a certain disease. Common uses of PCR include: 1. DNA cloning for sequencing (determining sequence of nucleotides) 2. Functional analysis of genes 3. Diagnosis of hereditary disease 4. Identification of genetic fingerprints (paternity testing/forensics) 5. Detection and diagnosis of infectious diseases The wide number of uses for PCR demonstrate just how broadly the scope of this technique spans. Procedure for Employing PCR: 1. Initiation--A small amount of the DNA containing the desired gene is placed in a test tube. Loose nucleotides that link into exact copies of the original gene are also added to the tube 2. Strand Separation--The two strands of the parent DNA are separated by heating the reaction environment (a solution) to 95-degrees Celsius for 15 seconds. This separation occurs when the hydrogen bonds between complimentary bases are disrupted by the heat. The product is a single-stranded DNA molecure, which is needed for the remainder of the PCR procedure. 3. Hybridization of Primers--Primers are added to the above solution, as they are needed to enable the polymerase chain reaction to occur. These primers are typically 20 to 30 nucleotides long. The solution is quickly cooled to 54-degrees Celsius, which allows each primer to hybridize to a DNA strand. One of the primers hybridizes to the 3' end of one strand, while the other primer hybridizes to the 3' end of the complementary strand. 4. DNA Synthesis--The solution is subsequently heated to 72-degrees Celsius, and DNA polymerase (often Taq, or Thermus Acquaticus, polymerase is used) comes in to elongate both primers in the direction of the target sequence. DNA synthesis takes place on both strands. These four steps (initiation, separation, hybridization and DNA synthesis) are repeated for 3 cycles, and by the third cycle, two short strands of only the target sequence are present. This means that from here out, amplification of only this target sequence (the one sought after) occurs exponentially. "The amplification is a millionfold after 20 cycles and a billionforld after 30 cycles, which can be carried out in less than an hour." PCR and Medical Diagnostics Of particualr importance (both in the past and presently) is the use of PCR in providing crucial diagnostic information about disease. Bacteria and viruses can be detected with the use of specific primers, and, perhaps most significantly, the presence of HIV can be discerned in people who have not yet shown symptoms or developed an immune response, meaning the HIV would otherwise be missed by traditional antibody assays. Moreover, PCR plays an important role in oncology, where the technique can be used both for early-detection and long-term monitoring of cancers. PCR tests can detect when cancerous cells have been killed, meaning treatment can be stopped, and can also detect relapse in cancer patients. References: Tymoczko, John L. "Biochemistry: A Short Course," second edition. pp 720-723. Freeman 2013. Smithsonian Institute Archives, 2004. "The History of PCR." Video courtesy of Garland Science Images courtesy of Gene Quantification